Analysis of a human DNA excision repair gene involved in group A xeroderma pigmentosum and containing a zinc-finger domain

Xeroderma pigmentosum (XP) is an autosomal recessive disease, characterized by a high incidence of sunlight-induced skin cancer. Cells from people with this condition are hypersensitive to ultraviolet because of a defect in DNA repair. There are nine genetic complementation groups of XP, groups A-H and a variant. We have cloned the mouse DNA repair gene that complements the defect of group A, the XPAC gene. Here we report molecular cloning of human and mouse XPAC complementary DNAs. Expression of XPAC cDNA confers ultraviolet-resistance on several group A cell lines, but not on lines of other XP groups. Almost all group A lines tested showed abnormality or absence of XPAC messenger RNAs. These results indicate that a defective XPAC gene causes group A XP. The human and mouse XPAC genes are located on chromosome 9q34.1 and chromosome 4C2, respectively. Human XPAC cDNA encodes a protein of 273 amino acids with a zinc-finger motif.     

Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases

Xeroderma pigmentosum is a monogenic disease characterized by hypersensitivity to ultraviolet light. The cells of xeroderma pigmentosum patients are defective in nucleotide excision repair, limiting their capacity to eliminate ultraviolet-induced DNA damage, and resulting in a strong predisposition to develop skin cancers. The use of rare cutting DNA endonucleases-such as homing endonucleases, also known as meganucleases-constitutes one possible strategy for repairing DNA lesions. Homing endonucleases have emerged as highly specific molecular scalpels that recognize and cleave DNA sites, promoting efficient homologous gene targeting through double-strand-break-induced homologous recombination. Here we describe two engineered heterodimeric derivatives of the homing endonuclease I-CreI, produced by a semi-rational approach. These two molecules-Amel3-Amel4 and Ini3-Ini4-cleave DNA from the human XPC gene (xeroderma pigmentosum group C), in vitro and in vivo. Crystal structures of the I-CreI variants complexed with intact and cleaved XPC target DNA suggest that the mechanism of DNA recognition and cleavage by the engineered homing endonucleases is similar to that of the wild-type I-CreI. Furthermore, these derivatives induced high levels of specific gene targeting in mammalian cells while displaying no obvious genotoxicity. Thus, homing endonucleases can be designed to recognize and cleave the DNA sequences of specific genes, opening up new possibilities for genome engineering and gene therapy in xeroderma pigmentosum patients whose illness can be treated ex vivo.     

The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta.

Xeroderma pigmentosum variant (XP-V) is an inherited disorder which is associated with increased incidence of sunlight-induced skin cancers. Unlike other xeroderma pigmentosum cells (belonging to groups XP-A to XP-G), XP-V cells carry out normal nucleotide-excision repair processes but are defective in their replication of ultraviolet-damaged DNA. It has been suspected for some time that the XPV gene encodes a protein that is involved in trans-lesion DNA synthesis, but the gene product has never been isolated. Using an improved cell-free assay for trans-lesion DNA synthesis, we have recently isolated a DNA polymerase from HeLa cells that continues replication on damaged DNA by bypassing ultraviolet-induced thymine dimers in XP-V cell extracts. Here we show that this polymerase is a human homologue of the yeast Rad30 protein, recently identified as DNA polymerase eta. This polymerase and yeast Rad30 are members of a family of damage-bypass replication proteins which comprises the Escherichia coli proteins UmuC and DinB and the yeast Rev1 protein. We found that all XP-V cells examined carry mutations in their DNA polymerase eta gene. Recombinant human DNA polymerase eta corrects the inability of XP-V cell extracts to carry out DNA replication by bypassing thymine dimers on damaged DNA. Together, these results indicate that DNA polymerase eta could be the XPV gene product.     

Yeast RAD14 and human xeroderma pigmentosum group A DNA-repair genes encode homologous proteins.

Xeroderma pigmentosum (XP), a human autosomal recessive disorder, is characterized by extreme sensitivity to sunlight and high incidence of skin cancers. XP cells are defective in the incision step of excision repair of DNA damaged by ultraviolet light. Cell fusion studies have defined seven XP complementation groups, XP-A to XP-G. Similar genetic complexity of excision repair is observed in the yeast Saccharomyces cerevisiae. Mutations in any one of five yeast genes, RAD1, RAD2, RAD3, RAD4, and RAD10, cause a total defect in incision and an extreme sensitivity to ultraviolet light. Here we report the characterization of the yeast RAD14 gene. The available rad14 point mutant is only moderately ultraviolet-sensitive, and it performs a substantial amount of incision of damaged DNA. Our studies with the rad14 deletion (delta) mutation indicate an absolute requirement of RAD14 in incision. RAD14 encodes a highly hydrophilic protein of 247 amino acids containing zinc-finger motifs, and it is similar to the protein encoded by the human XPAC gene that complements XP group A cell lines.     

Isolation of UV-resistant revertants from a xeroderma pigmentosum complementation group A cell line

Xeroderma pigmentosum (XP) is an autosomal recessive disease. Cells cultured from XP patients are hypersensitive to the lethal effects of UV light. Most XP cells are defective in an early stage in DNA repair of UV light-induced damage. The nature of the genetic defect of the XP syndrome has not been defined. To address this problem, we attempted to isolate UV-resistant cells from a cell line derived from an XP complementation group A (XPA) patient. By using a selection scheme capable of detecting one UV-resistant cell in a population of 10(8) cells, several UV-resistant clones were isolated at frequencies between 1 X 10(-7) and 2 X 10(-8). Here we describe the isolation and initial characterization of these phenotypic revertants.     

Molecular identification of a human DNA repair gene following DNA-mediated gene transfer

Although it has long been evident that the response of eukaryotes to DNA damaging agents is determined by the effectiveness of a variety of DNA repair systems, there is little detailed knowledge of the nature of these systems or the genes which control them. In humans, a number of hereditary conditions, including xeroderma pigmentosum, ataxia telangiectasia and Fanconi's anaemia, exhibit increased sensitivity to a variety of DNA damaging agents and a predisposition to cancer, suggesting a defect in some aspect of DNA repair. This report describes the identification of a human DNA repair gene following DNA-mediated gene transfer into Chinese hamster ovary (CHO) mutant cells, that like xeroderma pigmentosum cells, are sensitive to a variety of DNA damaging agents and are defective in the initial incision step of DNA repair. The resulting transformants exhibit normal resistance to DNA damaging agents and independent transformants demonstrate a common set of human DNA sequences associated with a human DNA repair gene. These observations provide the basis for the isolation and characterization of the human genes responsible for DNA repair.     

Expression cloning of a cocaine- and antidepressant-sensitive human noradrenaline transporter

At most synapses, chemical signalling is terminated by a rapid reaccumulation of neurotransmitter into presynaptic terminals. Uptake systems for the biogenic amines are the initial site of action for therapeutic antidepressants and drugs such as cocaine and the amphetamines. We have isolated a complementary DNA clone encoding a human noradrenaline transporter. The cDNA sequence predicts a protein of 617 amino acids, with 12-13 highly hydrophobic regions compatible with membrane-spanning domains. Expression of the cDNA clone in transfected HeLa cells indicates that noradrenaline transport activity is sodium-dependent and sensitive to selective noradrenaline transport inhibitors. Transporter RNA is localized to the brainstem and the adrenal gland. The predicted protein sequence demonstrates significant amino-acid identity with the Na+/gamma-aminobutyric acid transporter, thus identifying a new gene family for neurotransmitter transporter proteins. Analysis of its structure and function may lead to structure-based drug design for the treatment of human depression and could help determine whether transporter abnormalities underlie affective disorders.     

β-1,4-葡聚糖内切酶基因的克隆及在大肠杆菌中的表达

利用PCR方法从一株短小芽孢杆菌H9克隆了编码葡聚糖内切酶的基因（该序列已经被已收录于GeneBank,登录号为EF620915）,序列分析表明该基因读码框为1980bp,编码659个氨基酸。预测的酶由两个不连续的结构域组成,其一为N-端催化结构域,由糖基水解酶家族9组成,第二个结构域为C-端底物结合结构域,由碳水化合物绑定结构域家族3组成。将基因构建在大肠杆菌表达载体pET20b中得到重组质粒pET20b-EglA,将重组载体转化至大肠菌株BL21(DE3)菌株,基因在大肠杆菌中得到了良好的分泌表达, SDS-电泳图谱表明酶的分子大小约为73KD。     

人肥胖(obese)基因的人工合成及克隆

根据人肥胖基因的cDNA序列，通过合理的引物设计、链延伸反应、PCR反应以及分子克隆等步骤，成功地合成出编码瘦蛋白（Leptin）的肥胖基因（oB基因）全长片段，并将其克隆至PUc18载体质粒上．序列分析和酶切鉴定显示肥胖基因得到了正确合成和克隆．     

The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme

The RAD6 gene of the yeast Saccharomyces cerevisiae is required for a variety of cellular functions including DNA repair. The discovery that the RAD6 gene product can catalyse the covalent attachment of ubiquitin to other proteins suggests that the multiple functions of the RAD6 protein are mediated by its ubiquitin-conjugating activity.     

Molecular cloning of a new transforming gene from a chemically transformed human cell line

Molecular cloning of the transforming gene from a chemically transformed human osteosarcoma-derived cell line enables the gene to be mapped to chromosome 7 (7p11.4-7qter) and by this criterion and by direct hybridization to be shown to be unrelated to known oncogenes.